Proteinase inhibitor II (PIN2) is a serine proteinase inhibitor protein with two reactive sites, one inhibiting chymotrypsin and the other, trypsin (Bryant et al., 1976, Biochemistry 15: 3418–3424). PIN2 proteins are found in many Solanaceae plants including tomato (Gustafson and Ryan, 1976, J. Biol. Chem. 251: 7004–7010), potato (Bryant et al., 1976, Biochemistry 15: 3418–3424) and tobacco (Pearce et al., 1993, Plant Physiol. 102: 639–644). PIN2 proteins are present in leaves, flowers (Atkinson et al., 1993, Plant Cell 5: 203–213; Pena-Cortes et al., 1991, Plant Cell 3: 963–972; Brandstater et al., 1996, Mol. Gen. Genet. 252: 146–154), fruits (Pearce et al., 1988, Planta 175: 527–531), stems (Xu et al., 2001, Plant Mol. Biol. 47: 727–738) and tubers (Bryant et al., 1976, Biochemistry 15: 3418–3424).
Previously, plant proteinase inhibitors (PIs) were not known to inhibit endogenous plant proteases but were thought to have specificities for animal or microbial enzymes (Ryan, 1981, In The Biochemistry of Plants Vol.6 (Marcus, A., ed.). New York: Academic Press, pp. 351–370). The role of plant proteinase inhibitor proteins in defense is supported by studies of artificial insect diets and in vitro inhibition assays on insect gut proteases using purified proteinase inhibitor proteins from various plants (Hilder et al., 1993, Transgenic plants conferring insect tolerance: proteinase inhibitor approach. In: S. Kung and R. Wu (Eds.), Transgenic Plants, Volume 1, Academic Press, New York, pp. 317–338; Felton and Gatehouse, 1996, Antinutritive plant defence mechanisms. In: M. J. Lehane and P. F. Billingsley (Eds.), Biology Of The Insect Midgut, Chapman and Hall, London, pp. 373–416; Reeck et al., 1997, Proteinase inhibitors and resistance of transgenic plants to insects. In: N. Carozzi and M. Koziel (Eds.), Advances In Insect Control: The Role of Transgenic Plants, Taylor and Francis, London, pp. 157–183; Gatehouse, 1999, Biotechnological applications of plant genes in the production of insect-resistant crops. In: S. L. Clement and S. S. Quisenberry (Eds.), Global Plant Genetic Resources For Insect-resistant Crops, CRC Press, Boca Raton, pp. 263–280). Hence, the function of plant proteinase inhibitors was thought to be in the prevention of invasion through inhibition of foreign proteolytic enzymes of pests or pathogens (Ryan, 1989, BioEssays, 10: 20–24; Brzin and Kidric, 1995, Biotechnol. Genet. Eng. Rev. 13: 420–467).
There have been reports on the developmental regulation and tissue-specific accumulation of plant PIs (Rosahl et al., 1986, Mol. Gen. Genet. 202: 368–373; Sanchez-Serrano et al., 1986, Mol. Gen. Genet. 203: 15–20; Margossian et al., 1988, Proc Natl Acad Sci USA 85(21):8012–8016; Hendriks et al., 1991, Plant Mol. Biol. 17: 385–394; Pena-Cortes et al., 1991, Plant Cell, 3, 963–972; Lorberth et al., 1992, Plant J. 2: 477–486; Xu et al., 2001, Plant Mol. Biol. 47: 727–738). Proteinase inhibitor II from Solanum americanum (SaPIN2a) is highly expressed in the phloem and has possible involvement in regulating proteolysis in the sieve elements (Xu et al., 2001, Plant Mol. Biol. 47: 727–738). The localization of SaPIN2a mRNA and protein to the companion cells and sieve elements suggests regulation of proteolysis in phloem development/function.
What is needed are methods and compositions for expressing proteinase inhibitor proteins SaPIN2a and SaPIN2b by heterologous expression in transformed plants (i) to inhibit activities of endogenous proteases, particularly trypsin- and chymotrypsin-like activities; (ii) to delay the onset of senescence and/or programmed cell death, and/or increase their resistance to pests; and (3) for high level expression of proteinase inhibitor proteins for their isolation.